Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. Here, we targeted chicken ANP32A using CRISPR/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. We found that each chicken and human ANP32 family member had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.